JPS5971682A - Cultivation of euglena - Google Patents

Abstract

PURPOSE:To enable the mass cultivation of Euglena which assimilates fructose most easily among sacchrides, by culturing in a medium containing fructose-containing saccharide resource as a carbon source. CONSTITUTION:Euglena is devoid of an enzymatic system to assimilate monosaccharides and polysaccharides as carbon source, and has hitherto been cultured by using expensive ethanol, acetic acid, glutamic acid, etc. as carbon sources. A fructose-assimilable Euglena was obtained by separating Euglena glacilis 2K-14 strain from the field, and subjecting the strain to the adaptation to a fructose-containing medium. Accordingly, the strain can be cultured in a mass by using a fructose-containing sugar resource, such as the inexpensive carbon source by-produced from a sugar refining plant, e.g. black strap molasses, as the carbon source.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for culturing Euglena. Euglena is a protozoan that engages in photosynthesis, and its cell membrane is proteinaceous, so it is difficult to be digested by higher animal VL. The present inventor has reported that proteins have high nutritional value [Journal of the Japanese Society of Agricultural Chemistry, Vol. 51, No. 8 (1978)].

Dust may also contain many biologically active substances.

We expect it to be used in a variety of ways as a new material for biological work.

Although Euglena can be cultured autotrophically, industrial large-shake culture VL is not suitable in terms of cell yield and cost of lighting and carbon dioxide.

A lot of research has been done to grow Euglena heterotrophically, and it is used as a carbon source.

Ethanol, =xe,ylucose, glutamic acid, etc. have been used, but all of these are expensive. Therefore, in order to use Euklena industrially, it is necessary to establish a method that can achieve high yield and culture using an inexpensive carbon source. An object of the present invention is to provide a method for cultivating Euglena that can effectively utilize inexpensive shield resources produced as a by-product from sugar refineries.

Ding'S is characterized by culturing Euglena, which assimilates fructose among sugars, using fructose-containing shield resources as a carbon source.

According to the research of the present inventors, Euglena lacks an enzyme system that hydrolyzes and takes up polysaccharides, including invertase, outside the cells, and is unable to assimilate these saccharides. We also discovered that among monosaccharides, there are some Euglena strains that assimilate fructose better than glucose. Therefore, if this strain is isolated and adapted, Euglena can be effectively cultured using fructose as a carbon source, and as a result, Euglena can be cultivated in large quantities by effectively utilizing a carbohydrate source containing funotoctose.

The Euglena strain used in the present invention was obtained by culturing the Euglena strain in a medium containing fructose as the main carbon source and counting the number of cells. Cultivate, count the number of cells, compare the two, and find out whether it contains fructose? It can be obtained by selecting Euglena strains that grow better on the main carbon source. Of course, prior to this cultivation.

It can also be acclimatized by culturing in a medium containing Eugnale grape fructose as the main carbon source. Furthermore, IQ, the selected Euglena with excellent fructose assimilation ability was further acclimatized by repeating culture in a medium mainly containing fructose, and the fructose assimilation ability was determined. It can be increased in size.

Next, the concrete side will be shown.

Stocks preserved by the present inventor and tastes collected from the field.

A total of 46 strains were cultured 5 to 10 times in the following medium ■ containing fructose as the main carbon source and acclimatized.Medium ■Fructose 12ri/Nogluta ε
O37/Noaspartic acid
0.397 Noglycine 0.
39/Nohistidine salt - hydrate O, OS/i
Malic acid 6.597 tripotassium citric acid 0.5jJ/sodium nosuccinate hexahydrate 0.197 ammonium sulfate 0.25971 ammonium carbonate 0.259/potassium nolinic acid
1.25jE/magnesium carbonate
0.6/no calcium carbonate 0
．． 129/i Chinmonium sulfite O hexahydrate 501j+
9/i Manganese sulfate 18η/i Zinc sulfate/superhydrate 251nFI/It Molybdenum ammonium/tetrahydrate 41n9/Copper sulfate 1.2 nf! / ammonium nopanadate 0.5 m9 / Jl cobal sulfate heptahydrate 0.5 m9 / i boric acid
0.61ng/nickel sulfate/large hydrate I), 5 mfl/novitamin B, @hydrochloride 1myiii Hita S 7
B12 0.005 m9/no This strain was then treated with a culture medium and glycosylated with fructose (t).
The cells were cultured with shaking in the dark at an initial pH of 3 and 3 and a culture temperature of 27° C., respectively, and the number of cells was counted after reaching the second stationary phase using the same medium and the +it- medium I'.

Among the 46 strains tested, the following 6 strains assimilated fructose better than glucose.
The 3108 HB-10105 ZK-14 strain has a stronger ability to assimilate fructose than glucose and grows better).

This strain was grown in a medium containing fructoskeletal carbon ginseng for 20 hours.
After being subcultured for several months, the growth rate (cell number) was compared to the growth rate (cell number) in a medium with glucose as the sole carbon source under the same conditions.
The raw page size of 1.24 medium was shown.

Medium ■ Fructose 12 / i Milinmonium 59 / Magnesium sulfate, superhydride 0.5 g / Ammonium sulfite, large hydrate 50m97 Mancan sulfate 18 ■ / It Zinc sulfate, superhydride 2511 Ig / Nomolybdenum Ammonium acetate tetrahydrate 4m97 Copper sulfate 1.2MFl/i Ammonium vanadate 0.5π9/Jl
Cobalt sulfate superhydride 0.5 mfl/noboric acid 0.6 mkl/nickel nosulfate macrohydrate 0.5 m9/novitamin B1 hydrochloride 1M9/i vita i
y B, 2 0.005'R9/12
ZK-14 strain is Euglena gracilis (Eu, 5ri
It is a closely related strain of the two strains (ena, Fracl)isJ, and no difference in morphology from the two normal strains is observed under a microscope.

In the present invention, a Euglena strain that grows better when fructose is used as a carbon source than when glucose is used as a carbon source, in other words, a strain that has the ability to assimilate fructose rather than 5-glucose is used. In addition, many of the strains shown in Table 1 other than the ZK-14 strain, or those not shown in Table 1, have also been used for a long period of time. Acclimation increases the affinity for fructose and increases its assimilation ability, and these strains can also be used in the present invention if necessary. Euglena is treated with streptomycin.

Chloroplast-deficient strains r are given by known methods such as ultraviolet irradiation and high heat treatment, but like the ZK-14 strain, these strains can also be efficiently cultured on large plates in Q medium with fructose as the main carbon source. give.

The medium used contains fructose as the main carbon source, and also contains a nitrogen source, inorganic components, and vitamin B8. It is preferably included in vitamin B+2, and a representative example of this is medium ①.

Medium ■ (Use tap water as water; fufructose 12y/ammonium nophosphate 5y/) Magnesium sulfate.
Hydrate 0.59/i calcium carbonate
0.1. Ferrous sulfate/water treatment'@1m
9/I.

Vitamin Bl-@acid acid 2.5T
JIg/i vitamin BI2 5μ
Although fructose-containing glue resources containing other sugars can also be used in the present invention, Euglena.

Since it does not contain carbohydrases such as invertase, trisaccharides such as sucrose and other oligosaccharides and polysaccharides cannot be used as they are. Therefore, it is desirable to increase the amount of free fructose by cleaving the glycoside bonds of Cholera in advance using soap or a suitable enzyme. With sucrose-containing glue resources such as blackstrap molasses, for example, during heat sterilization, the medium 1 pH is made acidic to about 1.0 to 3.5, and after sterilization, the pH value is readjusted to a value suitable for culture, thereby easily achieving most of the objectives. sell. For more careful hydrolysis, use dilute acid at a temperature of 30 to 80°C for 30 to 12 minutes.
It only takes 2 hours. In addition, by using enzymes with specificity for each oligosaccharide and polysaccharide, it is possible to provide reaction conditions suitable for the action of each enzyme and perform a mild treatment. Using the obtained invertase.

In acetic acid solution at pH 5.4 and temperature 30°C, or in N
eurospora crassa, J: 'f)
invertase in acetate buffer at pH 7.5,
At a temperature of 38℃, the amount of free fructose reaches its maximum ILL fr
It is sufficient to carry out the enzymatic reaction until 'rw. Furthermore, when fructans such as inulin are used as a carbohydrate source, inulinase may be used, for example, in an acetate buffer, pH
5.0, treated at a temperature of 25''C.

Sugars are used as the main carbon source, and it is expected that free fructose and coexisting zero glucose will be dissolved or dissolved in the medium to make it optimal for the growth of Euglena. The concentration is 1.0 to 3. Ow
It is preferable to dilute it to 0 so that the amount is 100%.For dilution, it is appropriate to use 7K or wells of 7K, taking into account the replenishment of small amounts of gold.

Among the medium components other than the carbon source, ammonium salts, amino acids such as glutamic acid, or corn staple liquor are added as nitrogen sources. As ammonium salts, sulfur salts, 2-phosphate salts, hydrochlorides, etc. can all be used, but if the anion residue after nitrogen consumption is a problem, carbonate salts or ammonium salts can be used. Good results were obtained when using r. . Euglena is nitrate and nitrite.

urea? If a carbohydrate source such as blackstrap molasses is used and the effective crab component contained in this carbohydrate source is insufficient, the C/N ratio will be Add the above nitrogen source to match the growth conditions of Euglena.

Regarding inorganic substances other than nitrogen, when using invert sugar, fructan hydrolyzate, etc., a medium may be prepared according to the above-mentioned fructose medium. In the case of waste secret sugar, the ingredients are the raw sugar and the country of origin.

Although it varies greatly depending on the manufacturing method, etc., it generally contains most of the inorganic components necessary for culturing Euglena, and there is often no need to add them. However, magnesium salt and phosphoric acid are often in short supply, and in this case they must be replenished. Also.

Replenish other ingredients if they are insufficient.

In any case, it is preferable to supplement vitamins.
Add vitamin B1 and vitamin B1□ as above.

Carbohydrate sources with complex compositions, such as waste sugar, may contain substances that inhibit the growth of Euglena, but no such adverse effects have been observed in the range of tests conducted by the present inventors, and ordinary products do not. It seems safe to use. .

As described above, in the present invention, glaucoma sugars are used as a carbon source in the culture medium. The Euglena of the present invention has a particularly excellent ability to assimilate fructose, and is a carbohydrate source rich in fructose. For example, fructose, blackstrap molasses, waste molasses hydrolyzate, fructan hydrolyzate, and the like are particularly preferred.

The pH of the culture medium is suitably 2.5 to 8.0, typically 13.3 to 3.5. The culture temperature is 10
~3 (1'cρI is suitable, typically around 27℃,
fc is, for example, 26 to 28°C.

The illumination during culturing is in the range of 0 to io, ooo lux, and is generally 0. Obtained by the degree of illumination f
Since there may be differences in the content of physiologically active substances in LO cells, it is desirable to adjust the illumination as appropriate depending on the intended use of Euglena cells.

When using Euglena as a protein slurry, lighting is not particularly required, but if you want to produce bioactive substances such as vitamins C and E, unsaturated lipids, chlorophyll, and carotenoids.

500-20,000 lux light, typical tll
There is a light that emits white light. Also, when culturing the product on a specific plate, it may be more effective to irradiate it with blue light or red light.

When culturing, it is appropriate to use an aerated basin stone.

For example, io) aerate 2 to 5 air per minute for a culture medium. If the scale of the culture medium reaches a certain level, the capacity of the medium will increase accordingly. For certain products such as glycolic acid, it is desirable to vent oxygen gas through the VL.

Stirring is performed at 30 to 25 minutes per minute using an agitator or stirrer.
It is preferable to perform the rotation approximately 0 times. This is not preferable to the extent that the a-cells in culture do not settle, and violent agitation causes destruction of the cells.

Culture usually takes 2 to 6 days when cell division reaches the foot stage and the number of cells reaches its maximum. Q0 Euglena cells are collected by centrifugation and then dried by freeze drying, spray drying, hot air drying, etc. It can be dried to obtain a powdered product. The product has 8 different tones depending on the medium composition and reheating of the lighting.
Indicates yellowish, yellowish brown, greenish brown, pale brown, etc. For cell harvesting, if the culture conditions are selected so that the composition of the burial site is appropriate, the culture solution will be 1O~25, and the sugar yield will be 0.
．． 8-2% [range.

The chemical composition of the obtained Euglenar varies slightly depending on the medium composition and culture conditions. Table 2 shows representative examples of cases in which invert sugar was used as the carbon source, ammonium phosphate, and chloride source in the dark and under irradiation.

Table 2 (Dry 9, Medium) Ingredients Under darkness Under illumination Crude tannosilicate 48.4 55.0 Crude fat 12.0 15.2 Paramylon 30.6 17.6 Ash
Minutes 2.5 4.2 Others 6.5 8.9 Mifill Blackstrap molasses (filitubin-produced cane molasses) 250
Ammonium nophosphate 50y magnesium sulfate 2vitamin B,
0.25E Bitaban B12
, 50μyX way
A medium consisting of 10 ml of sulfuric acid
At H2.2, sterilization by autoclaving was carried out as usual.

After cooling, the pH was adjusted to 13.5 using sodium hydroxide and stirred at 100 revolutions per minute. I: ZK-14 strain was cultured at 28° C. in a 15-cell culture tank. Ventilation is done using air at a rate of 2 air per minute and VC lighting. When cultured for 3 days in near-dark conditions with only a little brightness from the endoscopic window, the pi dropped to 3.5.
The cell sum was 07 per liter.

Culture was stopped, cells were collected by centrifugation at 1,000 rprn, and lyophilized. The cells were obtained as an egg yolk powder, and the contents in the dry matter were as follows: O Crude protein 50% Paraepsilon 30% Crude fat 12% Ash 6 parts Vitamin CO, 05 parts Vitamin E 0.05 parts Also. The essential amino acid content in the protein was as follows (nitrogen 16
grams per gram).

Isoleucine 3,30 Isine 6.5 Lysine 5.8 Amino acid content 4.4 Aromatic 7.2 Threonine 4,4 Tryptophan 1.0 Palin 5.
3 Example 2 Blackstrap molasses (hokkaido sugar beet molasses) 2209
Glutamic acid 30f-Ammonium carbonate 201 Magnesium phosphate
105E Sumishi Calcium
5y Vitamin B10.1y Vitamin B, □ 50 μm tap water A medium consisting of 8i was adjusted to pH 2.0 with sulfuric acid overnight and left overnight at 40°C.
After adjusting the pH to 3.3 with an aqueous IJ solution and sterilizing it as usual, a chloroplast-deficient mutant of the ZK-14 strain obtained by pre-treating ZK-14 strain Euglena with streptomycin bacteria. The strains were exposed to a fluorescent light using the above medium in a 10i glass jar at 8000 lux on the outer surface.
Give 1:0 irradiation.

Cultured at 29°C. After 60 hours of culture, cells were collected,
It was dried by hot air drying method. Cell convergence was 200y. The product is light yellow-brown green in color.

The content in the dry matter is as follows! l).

Crude protein 52% Paramylon 28 Crude fat 11% Ash 5% Vitamin CO, 14 Vitamin E O 13% The essential amino acid composition in the protein was not significantly different from Example 1.

Example 3 Invert sugar (all sugar as dry matter) 120 Ammonium phosphate 50 Magnesium sulfate heptahydrate 5 Calcium carbonate
1y Ferrous sulfate 7X compound 0.0
A medium consisting of 19 vitamin Bl@hydrochloride, 0.025y vitamin BI2, 50 μm tap water and 10 μm was used. Among the components of this medium, ammonium phosphate is initially added to the medium at 20! As the culture progresses, additional doses are applied as the cells are consumed for a total of 50 y. This medium '1 was adjusted to pH 3.5.

Immediately, the cells were heat sterilized without prior hydrolysis, inoculated with Euglena ZK-14 strain, and cultured at 28° C. for 3 days in twilight. Dry cell yield was 160y. The product was Huangyan powder.

Crude protein 53% Paramylon 26 Crude fat 14% ash
min 2qb vitamin CO,07
Participant Vitaban E O, 13% Is this product? Make a feed with the following composition that uses protein as the only source, and feed 3
Breeding test was carried out using open-mouthed SD male white Nememi grapes. Dried Euglena cells 67% Vitamin mixture 1% Salt mixture 4th grade powdered cellulose 3% Choline paulate 0.3 thick soybean oil
5% Cornstarch Section 19.7 Again. As a comparative example, a similar feeding test was conducted using casein instead of dried Euglena cells.The feeding test was conducted for 4 weeks, and the growth was different when using Euglena as a whole protein source and when using Casein as a protein source. No difference was observed, nor was there any difference in examination of internal organs.

Claims (1)

[Claims] A method for culturing Euglena, which is characterized by culturing Euglena t, which assimilates fructose best among sugars, and a fructose-containing sugar resource as a carbon source.